PT1981858E - 3,5-di(aryl or heteroaryl)isoxazoles and 1,2,4-oxadiazoles as s1p1 receptor agonists, immunosuppresssive and anti-inflammatory agents - Google Patents

Abstract

The present invention relates to polycyclic compounds, processes for their production, their use as pharmaceuticals and to pharmaceutical compositions comprising them. X is -n= or -CH=; ring A is phenyl or pyridyl; R 1 is phenyl substituted by both haloalkyl and cycloalkyl.

Description

ΡΕ1981858 1

DESCRIPTION

" 3,5-D1 (ARIL OR HETEROARIL) ISOXAZOLES AND 1,2,4-OXADIOLS AS ANTAGONISTS OF S1P1 RECEPTORS, IMMUNOSUPRESSOR AND ΑΝΤΙ-INFLAMMATORY AGENTS " The present invention relates to polycyclic compounds, processes for their production, their use as pharmaceutical agents and pharmaceutical compositions containing them.

More specifically, the invention provides in a first aspect a compound of formula I

on what. X is -N = or = CH-;

Rx is biphenyl, 4-phenoxy-phenyl or 4- (phenyl-C1-4-alkoxy) -phenyl, wherein in each case at least one of the phenyl groups is monosubstituted by a halo-C1-4 alkyl; R2 is C1-4 alkyl optionally substituted by halogen, OH, NH2, C1-4 alkoxy or C1-4 alkylcarbonyloxy; amino; OH; C1-4 alkoxy; NH-OH; carboxy; sulfamoyl; carbamoyl; or NH-CO-C 1-4 alkyl; or R2 is R3-R4 -COOH or R3 -R4-CONH2 wherein R3 is SO2 -NH2, SO2 -2 SO2 -NH2, -NH2, NH2, NH3, or N (C1-4 alkyl) CO; and R 4 is C 1-6 alkylene, optionally interrupted by O, S or C = CH 2 or phenylene or C 3-6 cycloalkylene; and the ring A may be phenyl or pyridyl, each being optionally further substituted by halogen, C1-4 alkyl, haloC1-4 alkyl, C1-4 alkoxy, halo C1-4 alkoxy or nitrile; or R 2 is hydrogen with the proviso that ring A is 3-pyridyl; or a salt thereof. The halogen may be fluorine, chlorine or bromine, preferably fluorine or chlorine. The alkyl or alkoxy, as a group or present in a group, may be linear or branched. The C 1-6 alkylene may be straight or branched. C1-8 haloC1-8 alkyl or haloC1-8 alkoxy as a group or a part present in a group may be C1-8 alkyl or C1-8 alkoxy substituted by 1 to 5 halogens, for example CF3 or CF3 -CH2 -O-. C1-8 alkoxy-haloC1-8 alkoxy may be haloC1-8 alkoxy additionally substituted by C1-4 alkyl, for example at the 1-position. The same may apply to the other groups.

When R4 is phenylene or C3-6 cycloalkylene, it may be 1,4-phenylene or C3-6 cycloalkylene, for example cyclohexylene. The ring A may be optionally substituted by, for example, halogen, C1-4 alkyl, haloC1-4 alkyl, C1-4 alkoxy, haloC1-4 alkoxy or nitrile, preferably by halogen, C1-4 alkyl, haloC1-4 alkyl, C1-4 alkoxy or haloC1-4 alkoxy, more preferably by halogen C1-4 alkyl, haloC1-4 alkyl, or C1-4 alkoxy, and especially by halogen, C1-4 alkyl or C1-4 alkoxy. When ring A is pyridyl, it is preferably 3-pyridyl.

The following meanings are preferred independently, collectively or in any combination or sub-combination: i) R1 is biphenyl, 4-phenoxy-phenyl or 4- (phenyl-C1-4-alkoxy) -phenyl, wherein at least one of the groups phenyl has a haloC 1-4 alkyl, for example CF 3; iv) R2 is sulfamoyl; v) R 2 is R 3 -R 4 -COOH; or R3 -R4-CONH2, more preferably R3-R4-CONH2; vi) R 3 is SO 2 -NH; SO2 -N (C1-4 alkyl); NH-CO; or N (C 1-4 alkyl) CO; vii) R4 is C1-6 alkylene, and optionally interrupted by 0; viii) R2 is NH2; ix) Ring A is unsubstituted phenyl (i.e. without additional substituents other than R2). x) Ring A is substituted or unsubstituted pyridyl (i.e. without additional substituents other than R2).

The compounds of formula I may exist in their free form or in the salt form, for example, addition salts 4ΡE1981858 with, for example, organic and inorganic acids, for example, hydrochloric acid or acetic acid, or salts obtainable when R 2 is, or comprises COOH, with a base, for example alkali metal salts such as sodium or potassium, or substituted or unsubstituted ammonium salts.

It will be appreciated that the compounds of formula I may exist in the form of optical isomers, racemates or diastereoisomers. For example, R4 may comprise an asymmetric carbon atom when R4 is a branched alkylene. It is to be understood that the present invention includes all enantiomers and conformation isomers and mixtures thereof. Similar considerations apply to raw materials which exhibit asymmetric carbon atoms as mentioned above. The present invention also includes a process for the production of a compound of formula I, which process comprises

a) for the production of a compound of formula I, wherein X is -N = and R 2 is as previously defined, reacting a compound of formula II

Wherein the ring A and R2 are as previously defined, ΡΕ1981858

or a functional derivative thereof, for example, an ester, acyl chloride or anhydride activated with a compound of formula III

OH

wherein R 1 is as previously defined, or a functional derivative thereof; or b) for the production of a compound of formula I wherein X is CH and R 2 is NH 2 by reacting a compound of formula VI V '

wherein R 2 is as previously defined, with a compound of formula VII

wherein ring A is as defined above and R '2 is NH 2; or c) converting a compound of formula I into another compound of formula I, and recovering the resulting compound of formula I in free form or in the form of a salt, and when necessary converting a compound of formula I obtained in free form in the desired salt form or vice versa. 6 ΡΕ1981858

The steps of process a) to c) may be carried out according to methods known in the art, or as described below in the Examples.

Examples of the conversion of a compound of formula I into another compound of formula may include, for example, i) For the production of a compound of formula I wherein R1 is biphenyl, 4-phenoxy-phenyl or 4- (phenyl-C1-4 alkoxy) -phenyl, wherein at least one of the phenyl groups is mono-substituted, converting a compound of formula I wherein R 1 is not biphenyl, 4-phenoxy-phenyl or 4- (phenyl-C 1-4 -alkoxy) -phenyl, wherein at least one of the phenyl groups is monosubstituted, in a compound of formula I wherein R1 is biphenyl, 4-phenoxy-phenyl or 4- (phenyl-C1-4 alkoxy) -phenyl, wherein at least one of the phenyl groups is mono- substituted. ii) For the production of a compound of formula I, wherein R 2 is R 3 -R 4 -COOH, hydrolyzing a compound of formula I wherein the COOH present in R 2 is in the form of a physiologically hydrolyzable ester, for example methyl ester.

iii) For the production of a compound of formula I wherein X is -N = and R 2 is R 3 -R 4 -COOH, wherein R 3 is NH-CO or N (C 1-4 alkyl) CO and R 4 is as defined above, reacting a compound of formula I

wherein R1, X, R2 'and the ring A are as defined above, with an acylating agent. iv) For the production of a compound of formula I wherein R2 is R3 -R4-CONH2, reacting a compound of formula I, wherein R2 is R3 -R4 -COOH with an amidating agent.

The compound of formula III used as the starting material in process step a) can be obtained by reacting a compound of formula VIII R, -Sn

VIU wherein R 1 is as previously defined, with hydroxylamine.

The compound of formula VI used as the starting material in process step b) can be produced by reacting a compound of formula IX

R1 -COOH wherein R1 is such as hydroxylamine. previously defined, with 8 ΡΕ1981858

A compound of formula I wherein X is -N = and R 2 is NH 2 may also be produced by reacting a compound of formula III with a compound of formula II '

wherein ring A is as defined above, or a functional derivative thereof, for example an ester, acyl chloride or activated anhydride. The nitro group present in the resulting compound may then be reduced, for example, by catalytic hydrogenation.

Since the production of the starting materials is not particularly described, the compounds may be known or may be prepared analogously to methods known in the state of the art, or as described below.

The following Examples are descriptive of the invention.

Example 1: 4- [3- (2-Trifluoromethyl-biphenyl-4-yl) -isoxazol-5-yl] -phenylamine

9 ΡΕ1981858 a) 2-Trifluoromethyl-biphenyl-4-carbaldehyde oxime

Step a) To a solution of 4-cyano-2-trifluoroaniline (1eq) in benzene, n-pentylnitrite (1eq) was added under inert atmosphere at 50 ° C. After one hour at reflux, a second equivalent of n-pentylnitrite is added. After a further two hours of reflux, the reaction mixture is cooled to room temperature, and concentrated under reduced pressure. The dark residue is purified on silica gel using c-hexane - > c-hexane / ethyl acetate 9/1 as the mobile phase (pale orange oil).

Step b) The compound from step a) (1eq) is dissolved in formic acid (75%) and Ra-Ni (4eq) is added. After 3 hours at 80 ° C, the reaction mixture is filtered through Hyflo Super Cel® and the catalyst / Hyflo is washed 2 times with ethanol (carefully -> flammable). The resulting solution is concentrated (yield 61%, ester-acid mixture). This solution is used in step c) without further purification.

Step c) The compound of step b) and hydroxylamine hydrochloride (1.25 eq) are dissolved in ethanol, and K2CO3 (1.1 eq) is added. After 18 hours at ambient temperature, the reaction mixture is concentrated and after addition of water, it is extracted with ethyl acetate. Purification is achieved, 10Âμ1981858 after drying with Na2SO4 with silica gel, using c-hexane / ethyl acetate 9/1 as the mobile phase, resulting therefrom 2-trifluoromethyl-biphenyl-4-carbaldehyde oxime. ESI-MS (ESI +): 266 (M + 1H) +. b) 4- [3- (2-Trifluoromethyl-biphenyl-4-yl) -isoxazol-5-yl] -phenylamine

To a solution of 2-trifluoromethyl-biphenyl-4-carbaldehyde oxime (1eq) in CH2 Cl2 is added a 10% aqueous solution of NaOCl at 0 ° C. Thereafter, a solution of 4-ethynylaniline (1.1 eq) is added and the reaction mixture is then stirred at room temperature for 16 hours. The reaction mixture is diluted with CH 2 Cl 2 and extracted 3 times with water. The combined organic layers are dried over Na2 SO4 > 4, filtered and concentrated. Purification is achieved after drying on Na 2 SO 4 on silica gel using c-hexane / ethyl acetate 9/1/3 as the mobile phase. ESI-MS (ESI +): 381 (M + 1H) +.

Example 2: N- {4- [3- (2-Trifluoromethyl-biphenyl-4-yl) -isoxazol-5-yl] phenyl} succinamic acid

The compound (1eq) of Example 1 is dissolved in CH2 Cl2 and 4-methylmorpholine (2eq), and succinic anhydride (2eq) is added. After 16 hours at room temperature, the pure title product is obtained after filtration (white solid). ESI-MS (ESI +): 481 (M + 1H) +.

Example 3: N- {4- [3- (2-trifluoromethyl-biphenyl-4-yl) -isoxazol-5-yl] phenyl} succinamide

The compound of Example 2 is dissolved in DMF and subsequently N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC.HCl, 1.5 eq), hydroxybenzotriazole (HOBt; 1.3 eq), NH4 OH % in water (1.2 eq) and diisopropylethylamine (1.5 eq). After 16 hours at ambient temperature, the reaction mixture is concentrated and purified on silica gel (CH 2 Cl 2 / methanol 95/5 -> CH 2 Cl 2 / methanol / acetic acid 50% 90/10/0 as mobile phase), resulting in the title compound pure. ESI-MS (ESI +): 480 (Μ + 1H) +.

Example 4: 3- {4- [3- (2-Trifluoromethyl-biphenyl-4-yl) - [1,2,4] oxadiazol-5-yl] benzenesulfonylamino} propionic acid

12 ΡΕ1981858 a) N-hydroxy-2-trifluoromethyl-biphenyl-4-carboximide

Step a) To a solution of 4-cyano-2-trifluoroaniline (1eq) in benzene is added in an inert atmosphere, n-pentylnitrite (1eq), at 50 ° C. After one hour at reflux, a second equivalent of n-pentylnitrite is added. After a further two hours of reflux, the reaction mixture is cooled to room temperature and concentrated under reduced pressure. The dark residue is purified on silica gel using c-hexane -> c-hexane / ethyl acetate 9/1 as mobile phase (pale orange oil).

Step b) The compound from step a) is dissolved in THF, and hydroxylamine (50% in water, 10 eq) is added dropwise at -10 ° C. After 16 hours at ambient temperature, the reaction mixture is diluted with water and extracted with ethyl acetate. After drying the organic phase with Na2 SO4 > 4, purification is achieved on silica gel with c-hexane / ethyl acetate 75/25 as the mobile phase. b) 4- (2-Methoxycarbonyl-ethylsulfamoyl) -benzoic acid,

is obtained by reaction of 4-chlorosulfonyl-benzoic acid (1eq) with H-α-OMe-HCl in CH 2 Cl 2 using diisopropylethylamine (2eq) as the base. After 30 minutes at room temperature, the reaction mixture is extracted with water and the organic layer is dried over Na2SO4. Removal of solvent results in the title compound. c) 3- {4- [3- (2-Trifluoromethyl-biphenyl-4-yl) - [1,2,4] oxadiazol-5-yl] -benzenesulfonylamino} -propionic acid methyl ester 4- (2-Methoxycarbonyl (1eq) is dissolved in DMF, and EDC.HCl (1.3 eq) and HOBt (1.1 eq) are added. After 30 minutes at room temperature, N-hydroxy-2-trifluoromethyl-biphenyl-4-carboxamidine (1eq) is added and the reaction mixture is held at 90 ° C for 16 hours. After removal of the solvent, the residue is dissolved in ethyl acetate and extracted with saturated NaHCO3 solution. The title compound is obtained after drying the organic phase on Na 2 SO 4 on silica gel using 8/2/1 c-hexane / ethyl acetate / CH 2 Cl 2 as the mobile phase. ESI-MS (ESI +): 530 (Μ-1H). d) 3- {4- [3- (2-Trifluoromethyl-biphenyl-4-yl) - [1,2,4] -oxadiazol-5-yl] -benzenesulfonylamino} -propionic acid

14 ΡΕ1981858

LiOH (2 eq) is dissolved in methanol / water (1/1), and the ester (1 eq) is added. After 4 hours at 50 ° C, methanol is removed under reduced pressure, the pH is adjusted to ~ 3 with 1N HCl, and the reaction mixture is extracted 3 times with ethyl acetate. The combined organic phases are dried over Na 2 SO 4, filtered, concentrated and the title compound is obtained as a white powder after removal of the solvent. ESI-MS (ESI "): 516 (Μ-1H) ".

Example 5: 4- [3- (2-Trifluoromethyl-biphenyl-4-yl) - [1,2,4] oxadiazol-5-yl] -phenylamine

a) 5- (4-Nitro-phenyl) -3- (2-trifluoromethyl-biphenyl-4-yl] - [1,2,4] oxadiazole

It is obtained as a white powder using 4-nitrobenzoic acid instead of 4- (2-methoxycarbonyl-ethylsulfamoyl) -benzoic acid in the procedure of Example 1 - step c). ESI-MS (ESI +): 412 (M + 1H) +. B) The compound of step a) is dissolved in 1/1 methanol / ethyl acetate and hydrogenated at room temperature under normal pressure for 16 hours with Pd / C 20% as catalyst. After filtration through Hyflo Super Cel®, the reaction mixture is concentrated and purified on silica gel (CH 2 Cl 2 -> CH 2 Cl 2 / methanol 95/5 as the mobile phase), resulting in the title compound. ESI-MS (ESI +): 382 (M + 1H) +.

Example 6: 2,6-Dimethoxy-3- [3- (2-trifluoromethyl-biphenyl-4-yl) - [1,2,4] oxadiazol-5-yl] -pyridine

2,6-Dimethoxy-nicotinic acid (1eq) is dissolved in dioxane, and EDC.HCl (1.3eq) and HOBt (1.1eq) are added. After 30 minutes at room temperature, N-hydroxy-2-trifluoromethyl-biphenyl-4-carboxamidine (1eq) is added and the reaction mixture is held at 90 ° Celsius for 16 hours. After removal of the solvent, the residue is dissolved in ethyl acetate and extracted with saturated NaHCO3 solution. The title compound is obtained after drying the organic phase with Na2 SO4 on silica gel using c-hexane / ethyl acetate-α-hexane / ethyl acetate 100/23/30 as the mobile phase. ESI-MS (ESI): 426 (Μ-1H). 16 ΡΕ1981858

Following the procedure as described in the previous Examples, and using the appropriate starting materials, the compounds of formula I

wherein X, R1, R2 and ring A are as defined in Table 1 below. TABLE 1

Ex 2-CF 3 -4-biphenylyl 2-CF 3-4-biphenylyl CH 2 N-CO-CH 2 -C (CH 3) 2 -COOH phenyl CO-CH2-C (CH3) 2-CONH2 phenyl 508 (M + 1H) + 12 2-CF3-4-biphenylyl S02 -NH-CH2 -CH2 -CH2-CONH2 phenyl 515 (M-13) 2-CF3-4-biphenylyl N so2-nh2 phenyl 444 (M-1) 14 2-CF3-4-biphenylyl N-OH 383 (M-1) 15 2-CF 3 -4-biphenylyl N 3-CF 3 -phenyl 451 (M- -4-biphenylyl N -H-CO-CH 2 -CH 2 -COOH phenyl 480 (M-1) 17 2-CF 3 -4-biphenylyl 508 (M-1) 18 2-CF 3 -4-biphenylyl 479 (M-1H) 19 2-CF 3 -4-biphenylyl 507 (N-CO-CH 2 -C (CH 3) 2-CONH 2 (M + 1H) + 23 2-CF3-4-biphenylyl N-3-pyridyl 383 (M + 1H) + 2-CF3-4-biphenylyl biphenylyl N 3 -pyridyl 384 (M + 1H) + 26 2-CF 3 -4-biphenylyl N NH-OH 3-pyridyl 399 (M + 1H) + 27 2-CF 3 -4-biphenylyl N hn- cooh 3-pyridyl 483 (M + 1H) + 28 2-CF 3 -4-biphenylyl N coho 3-pyridyl 412 (M + 1H) + 2 2-CF 3-4-biphenylyl N 2 -pyridyl 412 (M + 1H) + 392 (M +

The compounds of formula I in free form or in pharmaceutically acceptable salt form exhibit valuable pharmaceutical properties, for example, as S1P1 receptor antagonists, for example as indicated in in vitro and in vivo tests, and are therefore indicated for therapy. A. In vitro

The compounds of formula I have binding affinity for individual human S1P1 receptors, as determined in the following assays: A. In vitro: GPCR activation assays, measuring GTP [γ-S] binding to membranes prepared from cells CHO expressing human EDG receptors

S1P1 (EDG-1) GTP binding assay [y-35S]: Homogenized membranes are prepared from CHO cell clones stably expressing a human EDG-1 with an N-terminal c-myc tag. Cells are grown in suspension in two 850 cm2 rotary vials for three to four days prior to harvesting. Cells are centrifuged, washed once with cold PBS, and resuspended in < 20 mL of Buffer A (20 mM HEPES, pH 7.4, 10 mM EDTA, complete protease inhibitor cocktail without EDTA [1 pellet / 25 mL]). The cell suspension is homogenized on ice using a Polytron homogenizer at 30,000 rpm, at three 15-second intervals each. The homogenate is first centrifuged at 2000 rpm in a low speed benchtop centrifuge, for 10 18 ΡΕ1981858 minutes. The supernatant, after passing through a cell filter, is then again centrifuged at 50,000 x g for 25 minutes at 4 ° C. The pellet is resuspended in Buffer B (15% glycerol, 20 mM HEPES, pH 7.4, 0.1 mM EDTA, complete protease inhibitor cocktail without EDTA [1 pellet / 10 mL]). The protein concentration of the preparation is determined using the BCA Protein Assay kit (Pierce) using BSA as standard. The membranes are aliquoted and stored frozen at -80 ° C. Solutions of test compounds in DMSO, in the range of 10 mM to 0.01 nM, are prepared. S1P is diluted in a 4% BSA solution as a positive control. The desired amount of membrane preparation is diluted with ice cold assay buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl 2, 0.1% fatty acid free BSA, 5 μM GDP) and well mixed with vortex 2 Âμl or less of compound are distributed in each well of a round bottomed 96-well polystyrene assay plate, followed by addition of 100 Âμl of diluted membranes (3-10 pg / well), and kept on ice until the addition of warm GTPyS. [35 S] -GTPyS is diluted 1: 1000 (v / v) with cold assay buffer and 100 μl is added to each well. The reaction is run at room temperature for 90 minutes before the membranes are collected onto a Unifilter® GF / B-96 Perkin Elmer filter plate using the Packard Filtermate Harvester. After several washings with wash buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl 2) and pass through 95% ethanol, the filter is dried in an oven at 37 ° C for 30 minutes. MicroScint-20 is added and the plate is sealed 19 ΡΕ1981858 for scintillation counting in TopCount. EC5o values are obtained by adjusting the GTP [γ-35S] binding curves (raw data) with the GraphPad Prism dose-response curve fitting tool. Six or twelve different concentrations are used to produce a concentration response curve (using three points for each concentration).

S1P binding assays 3, -4, -5 and -6 GTP [γ-j5S] are performed in a manner comparable to the S1P1 GTP [y-35S] binding assay using CHO cell membranes that stably express receptors without tag or c-terminal tag c-myc. For each membrane preparation, titration assays are performed first with the S1P control, to determine the optimal amount of membranes to be added to each test well.

The compounds of formula I are tested according to the above assay, and have been shown to exhibit selectivity for the S1P1 receptor. For example, the compounds of Examples 4 and 13 have an EC 50 < 1 μΜ in the above assay and are, for example, at least 20-fold more selective for S1P1 compared to S1P3, and for example at least 20-fold more selective for S1P1 compared to S1P5.

B. In vitro: FLIPR Calcium Flux Assay

The compounds of the invention are tested for antagonist activity on S1P1, S1P3, S1P5 and S1P6 with a 20 ΡΕ1981858 FLIPR calcium flow assay. Briefly, CHO cells expressing an S1P1 receptor are maintained in F-12K medium (ATCC), containing 5% FBS, with 500 pg / ml G418. Prior to assay, the cells are seeded in 384 clear bottom black plates at a density of 10,000 cells / well / 25 μl in the F-12K medium containing 1% FBS. On the second day, the cells are washed three times (25 æL each) with wash buffer. About 25 æl of dye is added to each well and the reaction is incubated for 1 hour at 37øC and 5% CO 2. Cells are then washed four times with wash buffer (25 æL each). Calcium flux is tested after adding 25 æl of SEW2871 solution (published by Rosen et al., Used as reference) to each well of cells. The same assay is performed with cells expressing each of the different S1P receptors. Titration in the FLIPR calcium flux assay is recorded over a 3 minute interval, and quantified as percentage response of the peak peak height relative to S1P-1 activation. The compounds of the invention are active in this assay at a concentration ranging from 10-12 to 3.10-5 nM. C. In vivo: Screening Assays for Measurement of Blood Lymphocyte Decrease 6-12

Measurement of circulating lymphocytes: The compounds to be tested are dissolved in DMSO / PEG200 and further diluted with deionized water. 1 mg / kg compound to be tested is administered to rats (Lewis strain, females, 21 ΡΕ1981858 weeks of age) in 4 ml / kg vehicle (max 2% DMSO / max. 2% PEG200 / water) via oral application . DMSO / PEG200 / water and FTY720 (0.3 mg / kg) are included as negative and positive controls, respectively.

Blood is collected from the sublingual vein 2, 6, 24 and 48 hours after administration under mild isoflurane anesthesia. Samples of whole blood are subjected to haematological analysis. Peripheral lymphocyte numbers are determined using an automated analyzer. Subpopulations of peripheral blood lymphocytes are stained with specific antibodies conjugated to fluorochromes and analyzed using a flow cytometer (Facscalibur). Two rats were used to determine the lymphocyte depleting activity of each compound tested. The result is given in ED50, which is defined as the effective dose required to result in a 50% decrease in blood lymphocytes. The compounds of formula I are tested according to the above assay and have an ED 50 of less than 10 mg / kg.

The compounds of formula I are therefore useful in the treatment and / or prevention of diseases or disorders mediated by interactions of lymphocytes, for example in transplantation, such as acute or chronic cell rejection, allografts or xenografts of tissues or organs or delayed graft function, graft versus host, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, thyroiditis of 22 ΡΕ1981858

Hashimoto, multiple sclerosis, myasthenia gravis, type I or II diabetes, and diseases associated therewith, vasculitis, pernicious anemia, Sjoegren's syndrome, uveitis, psoriasis, Graves ophthalmopathy, alopecia areata and others, allergic diseases such as asthma allergic contact dermatitis, allergic rhinitis / conjunctivitis, allergic contact dermatitis, inflammatory diseases, optionally with associated aberrant reactions, eg, inflammatory bowel disease, Crohn's disease or ulcerative colitis, intrinsic asthma, inflammatory lung injury, inflammatory liver injury, glomerular inflammatory, atherosclerosis, osteoarthritis, irritant contact dermatitis and other eczematous dermatitis, seborrheic dermatitis, cutaneous manifestations of immunologically mediated disorders, inflammatory eye disorders, keratoconjunctivitis, myocarditis or hepatitis, ischemic / reperfusion injury, for example , myocardial infarction, vascular accident rheumatoid arthritis, rheumatoid arthritis, cerebral rheumatoid arthritis, intestinal ischemia, renal failure or hemorrhagic shock, traumatic shock, cancer, e.g. breast cancer, T cell lymphomas or T cell leukemia, infectious diseases, e.g. toxic shock (e.g., superantigen induced), septic shock, adult respiratory distress syndrome or viral infections such as AIDS, viral hepatitis, chronic bacterial infection, or senile dementia. Examples of transplantation of solid cells, tissues or organs include, for example, pancreatic islets, stem cells, bone marrow, cornea, neuronal tissue, heart, lung, heart-lung, kidney, liver, intestine, pancreas, tracheal, or bladder 23 ΡΕ1981858 or esophagus. For the aforementioned uses, the dosage required will obviously vary depending on the mode of administration, the particular condition being treated and the effect desired.

In general, satisfactory results can be obtained systemically at daily dosages of about 0.03 to 5.0 mg / kg per body weight. A daily dosage indicated in a larger mammal, for example in humans, may range from about 0.5 mg to about 500 mg, conveniently administered, for example, in divided doses up to four times a day or in a delayed form. Suitable oral dosage form units comprise from about 0.1 to 50 mg of active ingredient.

The compounds of formula I may be administered by any conventional route, in particular enterally, for example orally, for example in the form of tablets or capsules, or parenterally, for example in the form of injectable solutions or suspensions, topically, for example in the form of lotions, gels, ointments or creams, or in a nasal or suppository form. Pharmaceutical compositions comprising a compound of formula I in free form or in the form of a pharmaceutically acceptable salt, in association with at least one pharmaceutically acceptable carrier or diluent, may be manufactured in conventional manner, by admixing with a pharmaceutically acceptable carrier or diluent. - 24 - ΡΕ1981858

The compounds of formula I may be administered in free form or in pharmaceutically acceptable salt form, for example, as indicated above. These salts may be prepared in the conventional manner and exhibit activities similar to those of the free compounds.

In accordance with the foregoing, the present invention further provides: A method for preventing or treating disorders or diseases mediated by lymphocytes, for example as indicated above, in a subject in need of such treatment, which method comprises administering of an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, to said subject; A method for preventing or treating chronic or acute rejection of T cell-mediated inflammatory or autoimmune transplants or diseases, for example as indicated above, in a subject in need of such treatment, which method comprises the administration of an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, to said subject; A compound of formula I, in free form or in a pharmaceutically acceptable salt form for use as a drug, for example, in any of the methods mentioned in 1.1 or 1.2 above. A pharmaceutical composition, for example, for use in any of the methods mentioned in 1.1 or 1.2 above, comprising a compound of formula I in free form or in pharmaceutically acceptable salt form, in association with a pharmaceutically acceptable diluent or carrier . A compound of formula I or a pharmaceutically acceptable salt thereof for use in the preparation of a pharmaceutical composition for use in any of the methods mentioned in 1.1 or 1.2 above.

The compounds of formula I may be administered as the sole active ingredient or as adjuvant together with, for example, other drugs, for example immunosuppressive or immunomodulatory agents, or other anti-inflammatory agents, for example for the treatment or prevention of rejection acute or chronic allograft or xenografts or autoimmune disorders, or as a chemotherapeutic agent, for example malignant cell anti-proliferation agent. For example, the compounds of formula I may be used in combination with a calcineurin inhibitor, for example cyclosporin A or FK 506; an inhibitor of mTOR, for example, rapamycin, 40-0- (2-hydroxyethyl) -rapamycin, CC1779, ABT578, AP23573, AP23464, AP23675, AP23841, TAFA-93, biolimus-7 or biolimus-9; a 26 ΡΕ1981858 ascomycin with immunosuppressive properties, for example, ABT-281, ASM981, etc .; corticosteroids; cyclophosphamide; azathioprine; methotrexate; leflunomide; mizoribine; mycophenolic acid or salt; mycophenolate mofetil; 15-deoxyspergualin or a homologue, analogue or immunosuppressive derivative thereof; a PKC inhibitor, for example as disclosed in WO 02/38561 or WO 03/82859, for example the compound of Example 56 or 70; an inhibitor of JAK3 kinase, for example N-benzyl-3,4-dihydroxy-benzylidene-cyanoacetamide Î ± -cyano- (3,4-dihydroxy) -N-benzylcinnamamide (Tyrphostin AG 490), prodigiosine 25-C (PNU156804), [4- (4'-Bromo-4'-hydroxyphenyl) -amino-6,7-dimethoxyquinazoline] (WHI-P131) P154), [4- (3 ', 5'-dibromo-4'-hydroxylphenyl) amino-6,7-dimethoxyquinazoline] WHI-P97, KRX-211, 3 - {(3R, 4R) -4- 3- [methyl- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -amino] -piperidin-1-yl} -3-oxo-propionitrile, in free form or in the form of a pharmaceutically acceptable salt , for example, monocytract (also referred to as CP-690,550), or a compound as described in WO 04/052359 or WO 05/066156; e.g., MHC, CD2, CD3, CD4, CD7, CD7, CD8, CD25, CD28, CD40, CD45, CD52, CD52, CD58, CD80, CD86 or their ligands; other immunomodulatory compounds, for example a recombinant binding molecule with at least a part of the extracellular domain of CTLA4 or a mutant thereof, for example at least one extracellular part of CTLA4 or a mutant thereof bound to a non-CTLA4 protein sequence , for example, CTLA41g (for example also referred to as ATCC 68629) or a mutant thereof, for example LEA29Y; inhibitors of adhesion molecules, for example LFA-1 antagonists, ICAM-1 or ICAM-3 antagonists, VCAM-4 antagonists or VLA-4 antagonists; or a chemotherapeutic agent, for example paclitaxel, gemcitabine, cisplatinum, doxorubicin or 5-fluorouracil; or an infectious agent.

When the compounds of formula I are administered together with other immunosuppressive / immunomodulatory, anti-inflammatory, chemotherapeutic or anti-infective agents, the dose of the immunosuppressive / immunomodulatory, anti-inflammatory, chemotherapy or anti-infective compound will of course vary depending on the type of co-drug used, for example, whether it is a steroid or a calcineurin inhibitor, the drug specifically used, the condition being treated, etc. According to the foregoing, the present invention further provides in a further aspect: A method as defined above comprising co-administering, for example concomitantly or in sequence, a non-toxic therapeutically effective amount of a compound of formula I and at least a second substance, for example an immunosuppressive, immunomodulatory, anti-inflammatory or chemotherapy drug, for example as indicated above. A pharmaceutical combination, for example a kit, comprising a) a first agent, which is a compound of formula I as disclosed herein, in free form or in the form of a pharmaceutically acceptable salt, and b) at least one co-agent, for example, an immunosuppressive, immunomodulatory, anti-inflammatory, chemotherapeutic and anti-infective agent. The kit may comprise instructions for its administration.

The terms " co-administration " or " combined administration " or the like as used herein includes administration of the selected therapeutic agents to an individual patient, and includes treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

The term " pharmaceutical combination " as used herein means a product which results from the admixture or combination of more than one active ingredient and includes fixed and non-fixed combinations of the active ingredients. The term " fixed combination " means that the active ingredients, for example a compound of formula I and a co-agent, are both administered to a patient simultaneously in the form of a single dosage or entity. The term " combination not fixed " means that the active ingredients, for example a compound of formula I and a co-agent, are both administered to a patient as separate entities either simultaneously or sequentially 29 ΡΕ1981858 without specific time limits, wherein this administration results in therapeutically effective levels of 2 compounds in the patient's body. The latter also applies to cocktail therapy, for example, the administration of 3 or more active ingredients.

Lisbon, September 1, 2010

Claims (6)

A compound of formula I on what. X is -N = or = CH-; R1 is biphenyl, 4-phenoxy-phenyl or 4- (phenyl-C1-4-alkoxy) -phenyl, wherein in each case at least one of the phenyl groups is monosubstituted by a halo-C1-4 alkyl; R2 is C1-4 alkyl optionally substituted by halogen, OH, NH2, C1-4 alkoxy or C1-4 alkylcarbonyloxy; amino; OH; C1-4 alkoxy; NH-OH; carboxy; sulfamoyl; carbamoyl; or HN-CO-C1-4alkyl; or R2 is R3-R4 -COOH or R3-R4-CONH2 wherein R3 is SO2 -NH, SO2 -N (C1-4alkyl), CO-NH, CON (C1-4alkyl), CH2-O, NH-CO, or N (C1-4 alkyl) CO; and R 4 is C 1-6 alkylene, optionally interrupted by O, S or C = CH 2 or phenylene or C 3-6 cycloalkylene; and the ring A may be phenyl or pyridyl, each being optionally further substituted by halogen, C1-4 alkyl, haloC1-4 alkyl, C1-4 alkoxy, halo C1-4 alkoxy or nitrile; or R 2 is hydrogen with the proviso that ring A is 3-pyridyl; or a salt thereof. A compound of formula I according to claim 1, selected from the group consisting of εΕ1981858 4- [3- (2-trifluoromethyl-biphenyl-4-yl) -isoxazol-5-yl] -phenylamine; N- {4- [3- (2-trifluoromethyl-biphenyl-4-yl) -isoxazol-5-yl] phenyl} succinamic acid; N- {4- [3- (2-trifluoromethyl-biphenyl-4-yl) -isoxazol-5-yl] phenyl} succinamide; 3- {4- [3- (2-Trifluoromethyl-biphenyl-4-yl) - [1,2,4] oxadiazol-5-yl] benzenesulfonylamino} propionic acid; 4- [3- (2-trifluoromethyl-biphenyl-4-yl) - [1,2,4] oxadiazol-5-yl] -phenylamine; 2,6-dimethoxy-3- [3- (2-trifluoromethyl-biphenyl-4-yl) - [1,2,4] oxadiazol-5-yl] -pyridine; and a compound of formula I according to claim 1, selected from the compounds included in the following table: Compound No. Compound No. 1 A-2-CF3-4-biphenylyl CH3 -CO2 -C (CH3) 2- COOH 2-CF 3 -4-biphenylyl 2-CF 3-4-biphenylyl 2-CF 3-4-biphenylyl 2-CF 3 -4-biphenylyl 2-CF 3-4-biphenylyl 2-CF 3-4-biphenylyl 2-CF 3-4-biphenylyl N 2 -CF 3 - COOH 2-CF 3 -4-biphenylyl 2-CF 3 -4-biphenylyl 2-CF 3 -4-biphenylyl 2 2-CF 3 -4-biphenylyl 2-CF 3 -4-biphenylyl NH 3-pyridyl 12 2-CF 3 -4-biphenylyl N 11-3-pyridyl 13 2-CF 3 -4-biphenylyl N 2-CF 3 -4-biphenylyl 2-CF 3 -4-biphenylyl 2-CF 3 -4-biphenylyl N 2-CF 3-4-biphenylyl N 2-CF 3 - 4-biphenylyl N COOH 3-pyridyl 17 2-CF 3 -4-biphenylyl N COOH 2-pyridyl 18 2-CF 3 -4-biphenylyl N ch 2 -NH 2 phenyl 3 ΡΕ1981858 A process for the preparation of a compound of formula I according to claim 1, which process comprises (i) for the preparation of a compound of formula I wherein X is -N = and R 2 is as previously defined , the reaction of a compound of formula II wherein the ring A and R 2 are as defined above, or a functional derivative thereof, for example an ester, acyl chloride or anhydride activated with a compound of formula III OH wherein R 1 is as defined above, or a functional derivative thereof; or ii) for the production of a compound of formula I wherein X is CH and R 2 is NH 2, reacting a compound of formula VI wherein R 1 is as defined above, with a compound of formula VII ## STR4 ## wherein ring A is as defined above and R '2 is NH 2; or iii) converting a compound of formula I into another compound of formula I, and recovering the resulting compound of formula I in free form or in the form of a salt, and when necessary converting a compound of formula I obtained in free form in the desired salt form or vice versa. A pharmaceutical composition comprising a compound of formula I according to claim 1 or 2, in free form or in the form of a pharmaceutically acceptable salt, in association with a pharmaceutically acceptable diluent or carrier. A pharmaceutical combination, for example a kit, comprising a) a first agent which is a compound of any one of claims 1 or 2, in free form or in the form of a pharmaceutically acceptable salt, and b) at least one compound for example an immunosuppressive, immunomodulatory, anti-inflammatory, chemo or anti-infective agent. Use of a pharmaceutical composition or compound according to any one of claims 1 or 5, in the manufacture of a medicament for the treatment and / or prevention of diseases or disorders mediated by lymphocyte interactions, for example in transplants and / or autoimmune diseases. Lisbon, September 1, 2010